S-nitrosylation (SNO) is a reversible protein modification that has the ability to alter the activity of target proteins. However, only a small number of SNO proteins have been found in the myocardium, and even fewer specific sites of SNO have been identified. Therefore, this study aims to characterize the myocardial S-nitrosothiol proteome and to determine the specific sites for S-nitrosothiol formation. We utilized a modified version of the SNO-resin assisted capture (SNO-RAC) technique in tandem with mass spectrometry. In brief, a modified biotin switch was performed using perfused mouse heart homogenates incubated with or without the S-nitrosylating agent S-nitrosoglutathione (GSNO). Our modified SNO-RAC protocol identified 1062 unique SNO proteins in GSNO-treated homogenates, many of which contained multiple SNO sites. These proteins represent the S-nitrosothiol proteome. Of the more than one thousand SNO proteins identified with GSNO treatment, only 121 showed constitutive SNO modification in non-GSNO treated homogenates. This study provides novel information regarding the myocardial S-nitrosothiol proteome, and yields additional information on the sites of S-nitrosothiol formation for many key proteins involved in myocardial energetics and contraction. Little is known with regard to the percentage of a given protein that is modified by SNO (i.e., SNO occupancy). Current methods allow for the relative quantification of SNO levels, but not for the determination of SNO occupancy. Objective: To develop a method for the measurement of SNO occupancy, and apply this methodology to determine SNO occupancy in the myocardium. We developed a differential cysteine-reactive tandem mass tag (cysTMT) labeling procedure for the measurement of SNO occupancy. To validate this cysTMT labeling method, we treated whole-heart homogenates with the S-nitrosylating agent S-nitrosoglutathione and determined maximal SNO occupancy. We also examined SNO occupancy under more physiological conditions and observed that SNO occupancy is low for most protein targets at baseline. Following ischemic preconditioning, SNO occupancy increased to an intermediate level compared to baseline and S-nitrosoglutathione treatment, and this is consistent with the ability of SNO to protect against cysteine oxidation. This novel cysTMT labeling approach provides a method for examining SNO occupancy in the myocardium. Using this approach, we demonstrated that IPC-induced SNO occupancy levels are sufficient to protect against excessive oxidation. We also performed studies to examine the role of nitric oxide in cardioprotection. Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Protein S-nitrosylation (SNO) is greatly increased following myocardial ischemic preconditioning (IPC) and it has been proposed that SNO may provide cardioprotection, in part, by reducing cysteine oxidation during ischemia-reperfusion (IR) injury. To test this hypothesis, we developed a new method to measure oxidation using resin assisted capture (RAC), similar to the SNO-RAC methods used in the quantification of S-nitrosylation. Langendorff perfused hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR) and homogenized. Each sample was divided into two equal aliquots, and a modified biotin switch was performed in order to simultaneously analyze SNO and oxidation. Using two independent measures, we identified 44 different proteins which showed increased S-nitrosylation with IPC. The majority (40) of these proteins also showed a decrease in cysteine oxidation following IR. We identified 53 cysteine residues from these 40 proteins that showed a decrease in oxidation at the same site of S-nitrosylation. These proteins included glyceraldehyde-3-phosphate dehydrogenase, electron transfer flavoprotein, and voltage-dependent anion-selective channel protein 2. Further, in an in vitro assay, oxidative challenge of purified alpha-ketoglutarate dehydrogenase increased the production of reactive oxygen species (ROS) by more than 60%. Interestingly, pretreatment with the S-nitrosylating agent S-nitrosoglutathione prevented the oxidation-induced increase in ROS production. These results suggest that SNO yields cardioprotective effects through two distinct mechanisms: (1) by directly blocking against the ROS-induced oxidation of cysteine residues, and (2) by reducing the production of ROS. In previous studies we have found that mitochondria are key regulators of preconditioning and most proteins showing an increase in SNO with IPC are mitochondrial. However, it is not clear how IPC transduces NO/SNO signaling to mitochondria. Using Langendorff perfused mouse hearts, we found that IPC-induced cardioprotection was blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor), ascorbic acid (a reducing agent to decompose SNO), or methyl-b-cyclodextrin (MbCD, a cholesterol sequestering agent to disrupt caveolae). IPC not only activated AKT/eNOS signaling but also led to translocation of eNOS to mitochondria. MbetaCD treatment disrupted caveolae structure, leading to dissociation of eNOS from caveolin-3 and blockade of IPC-induced activation of the AKT/eNOS signaling pathway. A significant increase in mitochondrial SNO was found in IPC hearts compared to perfusion control, and the disruption of caveolae by MbetaCD treatment not only abolished IPC-induced cardioprotection, but also blocked IPC-induced increase in SNO. In conclusion, these results suggest that caveolae transduce IPC-induced eNOS/NO/SNO acute cardioprotective signaling in the heart.